This invention relates to delivery of a bioactive agent. More particularly, the invention relates to a composition and method for delivering bioactive agents, such as DNA, RNA, oligonucleotides, proteins, peptides, and drugs, by facilitating their transmembrane transport or by enhancing their adhesion to biological surfaces. It relates particularly to a biodegradable cationic copolymer of a poly(alkylenimine) (PAI) and a hydrophilic polymer wherein the PAI and the hydrophilic polymer are covalently linked by a biodegradable linkage. The cationic copolymers of the present invention can be used in drug delivery and are especially useful for delivery of nucleic acids or any anionic bioactive agents.
Biodegradable polymers are gaining attention as drug delivery systems. R. Langer, New Methods of Drug delivery, 249 Science 1527-1533 (1990); B. Jeong et al., Biodegradable Block Copolymers as Injectable Drug-delivery Systems, 388 Nature 860-862 (1997). Delivering bioactive agents from a biodegradable delivery system is highly desirable because the need for a surgical procedure to remove the delivery system is avoided. Controlled release of bioactive agents can reduce the required frequency of administration by maintaining the concentration of the therapeutic agent at desired levels. One important means of maintaining the proper concentration is by controlling the degradation rate of the biodegradable drug delivery system.
Gene therapy is generally considered as a promising approach, not only for the treatment of diseases with genetic defects, but also in the development of strategies for treatment and prevention of chronic diseases such as cancer, cardiovascular disease and rheumatoid arthritis. However, nucleic acids, as well as other polyanionic substances, are rapidly degraded by nucleases and exhibit poor cellular uptake when delivered in aqueous solutions. Since early efforts to identify methods for delivery of nucleic acids in tissue culture cells in the mid 1950""s, steady progress has been made towards improving delivery of functional DNA, RNA, and antisense oligonucleotides in vitro and in vivo.
The gene carriers used so far include viral systems (retroviruses, adenoviruses, adeno-associated viruses, or herpes simplex viruses) or nonviral systems (liposomes, polymers, peptides, calcium phosphate precipitation and electroporation). Viral vectors have been shown to have high transfection efficiency when compared to non-viral vectors, but due to several drawbacks, such as targeting only dividing cells, random DNA insertion, their low capacity for carrying large sized therapeutic genes, risk of replication, and possible host immune reaction, their use in vivo is severely limited.
An ideal transfection reagent should exhibit a high level of transfection activity without needing any mechanical or physical manipulation of the cells or tissues. The reagent should be non-toxic, or minimally toxic, at the effective dose. It should also be biodegradable in order to avoid any long-term adverse side-effects on the treated cells. When gene carriers are used for delivery of nucleic acids in vivo, it is essential that the gene carriers themselves be nontoxic and that they degrade into non-toxic products. To minimize the toxicity of the intact gene carrier and its degradation products, the design of gene carriers needs to be based on naturally occurring metabolites.
Because of their sub-cellular size, nanoparticles are hypothesized to enhance interfacial cellular uptake, thus achieving in a true sense a local pharmacological drug effect. It is also hypothesized that there would be enhanced cellular uptake of drugs contained in nanoparticles (due to endocytosis) compared to the uptake of the corresponding free drug. Nanoparticles have been investigated as drug carrier systems for tumor localization of therapeutic agents in cancer therapy, for intracellular targeting (antiviral or antibacterial agents), for targeting to the reticuloendothelial system (parasitic infections), as an immunological adjuvant (by oral and subcutaneous routes), for ocular delivery with sustained drug action, and for prolonged systemic drug therapy.
As compared to viral gene carriers, there are several advantages to the use of non-viral based gene therapies, including their relative safety and low cost of manufacture. Non-viral gene delivery systems such as cationic polymers or synthetic gene carriers, e.g. poly-L-lysine (PLL), are being widely sought as alternatives and investigated intensively to circumvent some of the problems encountered with use of viral vectors. J. Cheng et al., Effect of Size and Serum Proteins on Transfection Efficiency of Poly((2-dimethylamino)ethyl methacrylate)-plasmid nanoparticles, 13 Pharm. Res. 1038-1042 (1996). There are several polymeric materials currently being investigated for use as gene carriers, of which poly-L-lysine (PLL) is the most popular, but few of them are biodegradable. Biodegradable polymers, such as polylactic/glycolic acid(negatively charged), and polylactide/glycolide(neutral) have been used as gene carriers in the form of non-soluble particulates. Amarucyama et al, Nanoparticle DNA Carrier with PLL Grafted Polysallanide Copolymer and Polylactic Acid, 8 Bioconjugate, 735-739(1997). In general, cationic polymers are known to be toxic and the PLL backbone is barely degraded under physiological conditions. It remains in cells and tissues and causes an undesirably high toxicity. A. Segouras and R. Dunlan, Methods for Evaluation of Biocompatibility of Synthetic Polymers, 1 J.Mater.Sci in Medicine, 61-68(1990).
PAIs such as poly(ethylenimine) (PEI) and polyspermine have been known as efficient gene carriers with high cationic charge potentials. Branched PEI consists of approximately 25, 50 and 25% of primary, secondary and tertiary amines and is able to condense and deliver DNA in vitro and in vivo, W. T. Godbey et al., 51 J. Biomed. Mater. Res. 321 (2000); W. T. Godbey et al., 60 J. Contr. Rel. 149 (1999); D. D. Dunlap et al., 25 Nucleic Acids Research 3095 (1997); O. Boussif et al., 92 Proc. Nat l Acad. Sci. USA 7297 (1995). Primary amines of PEI are reported to participate in forming complexes with DNA by ionic interaction with phosphate groups, while the secondary and tertiary amines cause a substantial endosomal disruption after endocytosis due to their buffering effect which contributes to the high transfection efficiency of PEI. The high transfection efficiency of PEI, along with its cytotoxicity, strongly depends on its molecular weight. It is generally believed that PEI with a molecular weight higher than 25 K displays a high transfection efficiency and toxicity, while PEI with molecular weight less than 1.8 K shows almost no transfection, but is less toxic, S. Brunner et al., 7 Gene Ther. 401 (2000); D. Fischer et al., 16 Pharm. Res. 1273 (1999); W. T. Godbey et al., 45 J. Biomed. Mater. Res. 268 (1999). In addition, just like most cationic polymers, PEI has drawbacks since complexes of PEI and DNA are often poorly soluble under physiological conditions, A. V. Kabanov et al., 6 Bioconjugate Chem. 7 (1995).
Di-block and graft copolymers of PEI and PEG have been synthesized and investigated by several research groups, Y. Akiyama et al., 33 Macromolecules 5841 (2000); S. V. Vinogradov et al., 9 Bioconjugate Chem. 805 (1998). Although copolymers of high molecular weight PEI and PEG exhibit considerable transfection efficiency, with the employment of high molecular weight PEI, cytotoxicity still remains as a problem. In addition, none of the existing copolymers of PEI and PEG are biodegradable.
In view of the foregoing, development of a gene carrier for gene therapy and drug delivery that is non-toxic, biodegradable, and capable of forming nanoparticles, or transfection complexes will be appreciated and desired. The novel gene carrier of the present invention comprises a novel cationic copolymer of a poly(alkylenimine) (PAI) and a hydrophilic polymer, wherein the PAI and the hydrophilic polymer are covalently linked by a biodegradable linkage. The biodegradable cationic copolymer of the present invention is useful for drug delivery, especially for delivery of nucleic acids, other anionic bioactive molecules, or both, and is readily susceptible to metabolic degradation after incorporation into the cell.
The present invention provides a biodegradable cationic copolymer, having reduced in vivo and in vitro toxicity, useful for delivery of drugs or other bioactive agents to an individual in need thereof.
The present invention also provides biodegradable water soluble cationic copolymers that are able to condense DNA and form stable complexes with DNA under physiological conditions.
The present invention further provides an efficient non-viral polymer-based water-soluble system for delivery of DNA or RNA to a target cell.
The present invention further provides an efficient polymer-based water-insoluble system for delivery of proteins or other bioactive agents.
The biodegradable cationic copolymer of the present invention comprises a biodegradable cationic copolymer of a poly(alkylenimine) (PAI) and a hydrophilic polymer wherein the PAI and the hydrophilic polymer are covalently linked by a biodegradable linkage. Preferably, the hydrophilic polymer is a member selected from the group consisting of polyethylene glycol (PEG), poloxamers, poly(acrylic acid), poly(styrene sulfonate), carboxymethylcellulose, poly(vinyl alcohol), polyvinylpyrrolidone, alpha-substituted poly(oxyalkyl) glycols, poly(oxyalkyl) glycol copolymers and block copolymers, and activated derivatives thereof. More preferably, the hydrophilic polymer is a member selected from the group consisting of polyethylene glycol (PEG), poloxamers, poly(acrylic acid), poly(styrene sulfonate), carboxymethylcellulose, poly(vinyl alcohol) and polyvinylpyrrolidone. The most preferred hydrophilic polymer is polyethylene glycol (PEG). Preferably, the average molecular weight of the PAI is within a range of 600 to 100,000 Daltons and the average molecular weight of the hydrophilic polymer is within a range of 500 to 20,000 Daltons. The PAI is conjugated to the hydrophilic polymer by a biodegradable linkage which can be an ester, amide or urethane, depending on the required degradation rate. The molar ratio of the PAI to the hydrophilic polymer is preferably within a range of 0.1 to 2. Due to the multi-functionality of PAIs, the solubility in water of the synthesized copolymers can be controlled by the reaction conditions. A preferred cationic copolymer is a copolymer of a low molecular weight PAI and PEG, which exhibits negligible toxicity and high transfection efficiency.
Hydrophilic PEG is expected to reduce the toxicity of the copolymer, improve the poor solubility of the PAI and DNA complexes, and help to introduce biodegradable groups by reaction with the primary amines in the PAI. Considering the dependence of transfection efficiency and cytotoxicity on the molecular weight of the PAI, high transfection efficiency is expected from an increased molecular weight of the copolymer and low cytotoxicity from the degradation of the copolymer into minimally toxic low molecular weight PAIs.
The biodegradable copolymers can be synthesized by relatively simple and inexpensive methods. The biodegradable water soluble cationic copolymer is synthesized by reacting a branched or linear PAI with PEG, having difunctional groups, that produces biodegradable linkages with the primary amino groups in the PAI. Initial polymer concentrations of the reaction mixture, the number of functional groups in the PAI which is related to the initial molecular weight of the PAI, and the other reaction conditions should be carefully controlled to prevent possible cross-linking reactions. The molecular weight of the copolymer and the molar ratio of the PAI and PEG in the copolymer can be adjusted by changing the initial concentration of the PAI and the difunctional PEG. The biodegradable water insoluble copolymer can be synthesized using a method similar to that employed in making the water soluble copolymers except for the higher initial concentration of the reaction mixture and the higher molecular weight of the initial PAI.
The cationic copolymers of the present invention can spontaneously form discrete nanometer-sized particles with a nucleic acid, which can promote more efficient gene transfection into mammalian cells and show reduced cell toxicity. The copolymer of the present invention is readily susceptible to metabolic degradation after incorporation into animal cells. Moreover, the water soluble cationic copolymer can form an aqueous micellar solution which is particularly useful for systemic delivery of various bioactive agents such as DNA, proteins, hydrophobic or hydrophilic drugs. The water insoluble copolymer can form cationic nanoparticles which is particularly useful for local drug delivery. Therefore, the biocompatible and biodegradable cationic copolymer of this invention provides an improved gene carrier for use as a general reagent for transfection of mammalian cells, and for the in vivo application of gene therapy.
The present invention further provides transfection formulations, comprising a novel cationic copolymer complexed with a selected nucleic acid, in the proper charge ratio (positive charge of the copolymer/negative charge of the nucleic acid), that is optimally effective for both in vivo and in vitro transfection. Particularly, the weight ratio of DNA to the cationic copolymer is preferably within a range of 1:0.3 to 1:16.
This invention also provides for a method of transfecting a cell in vitro with biodegradable water soluble cationic copolymers and a selected plasmid DNA, comprising the steps of:
(a) providing a composition comprising a complex with an effective amount of positively charged cationic biodegradable polymer and plasmid DNA.
(b) Contacting the cell with an effective amount of the composition such that the cell internalizes the selected plasmid DNA; and
(c) Culturing the cell with the internalized selected plasmid DNA under conditions favorable for the growth thereof.